Composition for sustained release of human growth hormone

ABSTRACT

A composition, and methods of forming and using said composition, for the sustained release of biologically active, stabilized human growth hormone (hGH). The sustained release composition of this invention comprises a polymeric matrix of a biocompatible polymer and particles of biologically active, stabilized hGH, wherein said particles are dispersed within the biocompatible polymer. The method of the invention for producing a composition for the sustained release of biologically active hGH, includes dissolving a biocompatible polymer in a polymer solvent to form a polymer solution, dispersing particles of biologically active, stabilized hGH in the polymer solution, and then solidifying the polymer to form a polymeric matrix containing a dispersion of said hGH particles. The method for using a composition of the invention is a method for providing a therapeutically effective blood level of biologically active, non-aggregated hGH in a subject for a sustained period. In this method, a subject is administered an effective dose of the sustained release composition of the present invention. The method of using the sustained release composition of the present invention comprises providing a therapeutically effective blood level of biologically active, non-aggregated human growth hormone in a subject for a sustained period by administering to the subject a dose of said sustained release composition.

RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 07/984,323, filed Dec. 2, 1992, now abandoned, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Human growth hormone (hGH) is a protein secreted by the pituitary glandand which can be produced by recombinant genetic engineering. hGH willcause growth in all bodily tissues which are capable of growth.

hGH is typically used to treat patients suffering from hypopituitarydwarfism. Currently, aqueous hGH is administered as a subcutaneous bolusthree times a week or once daily to patients to maintain suitable serumlevels of hGH. For patients chronically receiving hGH, the frequentinjections result in patient compliance problems.

To resolve the problems associated with repetitive injections of aqueoushGH, attempts have been made to formulate controlled release devicescontaining higher doses of hGH than a bolus injection, encapsulatedwithin a polymeric matrix wherein the hGH would be released in vivo overa period of about a week or more.

However, these controlled release devices often exhibited high initialbursts of hGH release and minimal hGH release thereafter. Further, dueto the high concentration of hGH within these controlled releasedevices, the hGH molecules have tended to aggregate after several daysto form aggregated hGH which is immunogenic in vivo and likely hasreduced biological activity.

Therefore, a need exists for a means for sustaining the release ofbiologically active hGH in vivo without causing an immune systemresponse over the release period of the hGH.

SUMMARY OF THE INVENTION

This invention relates to a composition, and methods of forming andusing said composition, for the sustained release of biologicallyactive, stabilized human growth hormone (hGH). The sustained releasecomposition of this invention comprises a polymeric matrix of abiocompatible polymer and particles of biologically active, stabilizedhGH, wherein said particles are dispersed within the biocompatiblepolymer.

The method of the invention for forming a composition for the sustainedrelease of hGH, includes dissolving a biocompatible polymer in a polymersolvent to form a polymer solution, dispersing particles of biologicallyactive, stabilized hGH in the polymer solution, and then solidifying thepolymer to form a polymeric matrix containing a dispersion of said hGHparticles.

The method of using the sustained release composition of the presentinvention comprises providing a therapeutically effective blood level ofbiologically active, non-aggregated human growth hormone in a subjectfor a sustained period by administering to the subject a dose of saidsustained release composition.

The advantages of this sustained release formulation for hGH includelonger, more consistent in vivo blood levels of hGH, lower initialbursts of hGH, and increased therapeutic benefits by eliminatingfluctuations in serum hGH levels. The advantages also include increasedpatient compliance and acceptance by reducing the required number ofinjections. The advantages further include the ability to use smalleramounts of hGH compared to bolus injection regimen because serum hGHlevels are maintained closer to therapeutical thresholds.

DETAILED DESCRIPTION OF THE INVENTION

The human growth hormone (hGH) used in this invention is biologicallyactive hGH in its molecular (monomeric or non-aggregated) form.Molecular hGH is typically non-immunogenic.

Aggregated hGH may induce an immune response resulting in antibodiesformed against hGH. This may compromise the efficacy of long-term hGHtherapy. Additionally, aggregated hGH may stimulate an auto-immuneresponse to endogenous hGH.

A sustained release of biologically active, non-aggregated human growthhormone is a release which results in measurable serum levels ofbiologically active, monomeric hGH over a period longer than thatobtained following direct administration of aqueous hGH. It is preferredthat a sustained release be a release of hGH for a period of about aweek or more, and more preferably for a period of about two weeks ormore.

A sustained release of biologically active, non-aggregated hGH from apolymeric matrix can be continuous or non-continuous release withrelatively constant or varying rates of release. The continuity of hGHreleased and level of hGH released can be established by using,inter-alia, one or more types of polymer compositions, hGH loadings,and/or selection of excipients to produce the desired effect.

Stabilized (hGH) comprises biologically active, non-aggregated hGH whichis complexed with at least one type of multivalent metal cation, havinga valency of +2 or more, from a metal cation component. Stabilized hGHin the sustained release composition of the present invention is inparticulate form.

Suitable multivalent metal cations include metal cations contained inbiocompatible metal cation components. A metal cation component isbiocompatible if the cation component is non-toxic to the recipient, inthe quantities used, and also presents no significant deleterious oruntoward effects on the recipient's body, such as an immunologicalreaction at the injection site.

Typically, the molar ratio of metal cation component to hGH, for themetal cation stabilizing the hGH, is between about 4:1 to about 10:1.

A preferred metal cation used to stabilize hGH is Zn⁺². In a morepreferred embodiment, the molar ratio of metal cation component,containing Zn⁺² cations, to hGH is about 6:1.

The suitability of a metal cation for stabilizing hGH can be determinedby one of ordinary skill in the art by performing a variety of stabilityindicating techniques such as polyacrylamide gel electrophoresis,isoelectric focusing, reverse phase chromatography, HPLC and potencytests on hGH lyophilized particles containing metal cations to determinethe potency of the hGH after lyophilization and for the duration ofrelease from microparticles. In stabilized hGH, the tendency of hGH toaggregate within a microparticle during hydration in vivo and/or to losebiological activity or potency due to hydration or due to the process offorming a sustained release composition, or due to the chemicalcharacteristics of a controlled release composition, is reduced bycomplexing at least one type of metal cation with hGH prior contactingthe hGH with a polymer solution.

Stabilized hGH is typically stabilized against significant aggregationin vivo over the sustained release period.

Stabilized hGH is typically stabilized against significant aggregationin vivo over the sustained release period. Significant aggregation isdefined as an amount of aggregation resulting in aggregation of about15% or more of the initial amount of encapsulated hGH monomer.Preferably, aggregation is maintained below about 5% of the initial doseof hGH monomer. More preferably, aggregation is maintained below about2% of the initial dose.

The hGH in a hGH sustained release composition can also be mixed withother excipients, such as bulking agents or additional stabilizingagents, such as buffers to stabilize the hGH during lyophilization.

Bulking agents typically comprise inert materials. Suitable bulkingagents are known to those skilled in the art.

A polymer, or polymeric matrix, suitable for the sustained releasecomposition of the present invention must be biocompatible. A polymer isbiocompatible if the polymer, and any degradation products of thepolymer, are non-toxic to the recipient and also present no significantdeleterious or untoward effects on the recipient's body, such as animmunological reaction at the injection site.

The polymer of the hGH sustained release composition must also bebidegradable. Biodegradable as defined herein, means the compositionwill degrade or erode in vivo to form smaller chemical species.Degradation can result, for example, by enzymatic, chemical and physicalprocesses. Suitable biocompatible, biodegradable polymers include, forexample, poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polycyanoacrylates,poly(p-dioxanone), poly(alkylene oxalate)s, biodegradable polyurethanes,blends and copolymers thereof.

Further, the terminal functionalities of the polymer can be modified.For example, polyesters can be blocked, unblocked or a blend of blockedand unblocked polymers. A blocked polymer is as classically defined inthe art, specifically having blocked carboxyl end groups. Generally, theblocking group is derived from the initiator of the polymerization andis typically an alkyl group. An unblocked polymer is as classicallydefined in the art, specifically having free carboxyl end groups.

Acceptable molecular weights for polymers used in this invention can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as the desired polymer degradation rate,physical properties such as mechanical strength, and rate of dissolutionof polymer in solvent. Typically, an acceptable range of molecularweights is of about 2,000 Daltons to about 2,000,000 Daltons. In apreferred embodiment, the polymer is a biodegradable polymer orcopolymer. In a more preferred embodiment, the polymer is apoly(lactide-co-glycolide) (hereinafter "PLGA") with a lactide:glycolideratio of about 1:1 and a molecular weight of about 5,000 Daltons toabout 70,000 Daltons. In an even more preferred embodiment, themolecular weight of the PLGA used in the present invention has amolecular weight of about 6,000 to about 31,000 Daltons.

The amount of hGH, which is contained in a dose of sustained releasemicroparticles, or in an alternate sustained release device, containingbiologically active, stabilized hGH particles is a therapeutically orprophylactically effective amount, which can be determined by a personof ordinary skill in the art taking into consideration factors such asbody weight, condition to be treated, type of polymer used, and releaserate from the polymer.

In one embodiment, an hGH sustained release composition contains fromabout 0.01% (w/w) to about 50% (w/w) of biologically active, stabilizedhGH particles. The amount of such hGH particles used will vary dependingupon the desired effect of the hGH, the planned release levels, thetimes at which hGH should be released, and the time span over which thehGH will be released. A preferred range of hGH particle loading isbetween about 0.1% (w/w) to about 30% (w/w) hGH particles. A morepreferred range of hGH particle loading is between about 0.1% (w/w) toabout 20% (w/w) hGH particles. The most preferred loading of thebiologically active, stabilized hGH particles is about 15% (w/w).

In another embodiment, a hGH sustained release composition also containsa second metal cation component, which is not contained in thestabilized hGH particles, and which is dispersed within the polymer. Thesecond metal cation component preferably contains the same species ofmetal cation, as is contained in the stabilized hGH. Alternately, thesecond metal cation component can contain one or more different speciesof metal cation.

The second metal cation component acts to modulate the release of thehGH from the polymeric matrix of the sustained release composition, suchas by acting as a reservoir of metal cations to further lengthen theperiod of time over which the hGH is stabilized by a matal cation toenhance the stability of hGH in the composition.

A metal cation component used in modulating release typically containsat least one type of multivalent metal cation. Examples of second metalcation components suitable to modulate hGH release, include, or contain,for instance, Mg(OH)₂, MgCO₃ (such as 4MgCO₃.Mg(OH)₂.5H₂ O), ZnCO₃ (suchas 3Zn(OH)₂.2ZnCO₃), CaCO₃, Zn₃ (C₆ H₅ O₇)₂, Mg(OAc)₂, MgSO₄, Zn(OAc)₂,ZnSO₄, ZnCl₂, MgCl₂ and Mg₃ (C₆ H₅ O₇)₂. A suitable ratio of secondmetal cation component-to-polymer is between about 1:99 to about 1:2 byweight. The optimum ratio depends upon the polymer and the second metalcation component utilized.

A polymeric matrix containing a dispersed metal cation component tomodulate the release of a biologically active agent from the polymericmatrix is further described in co-pending U.S. patent application Ser.No. 08/237,057, filed May 3, 1994, and co-pending PCT Patent ApplicationPCT/US95/05511, the teachings of which are incorporated herein byreference in their entirety.

The hGH sustained release composition of this invention can be formedinto many shapes such as a film, a pellet, a cylinder, a disc or amicroparticle. A microparticle, as defined herein, comprises a polymericcomponent having a diameter of less than about one millimeter and havingstabilized hGH particles dispersed therein. A microparticle can have aspherical, non-spherical or irregular shape. It is preferred that amicroparticle be a microsphere. Typically, the microparticle will be ofa size suitable for injection. A preferred size range for microparticlesis from about 1 to about 180 microns in diameter.

In the method of this invention for forming a composition for thesustained release of biologically active, non-aggregated hGH, a suitableamount of particles of biologically active, stabilized hGH are dispersedin a polymer solution.

A suitable polymer solution contains between about 1% (w/w) and about30% (w/w) of a suitable biocompatible polymer, wherein the biocompatiblepolymer is typically dissolved in a suitable polymer solvent.Preferably, a polymer solution contains about 2% (w/v) to about 20%(w/v) polymer. A polymer solution containing 5% to about 10% (w/w)polymer is most preferred.

A suitable polymer solvent, as defined herein, is solvent in which thepolymer is soluble but in which the stabilized hGH particles aresubstantially insoluble and non-reactive. Examples of suitable polymersolvents include polar organic liquids, such as methylene chloride,chloroform, ethyl acetate and acetone.

To prepare biologically active, stabilized hGH particles, hGH is mixedin a suitable aqueous solvent with at least one suitable metal cationcomponent under pH conditions suitable for forming a complex of metalcation and hGH.

Suitable pH conditions to form a complex of hGH typically include pHvalues between about 7.0 and about 7.4. Suitable pH conditions aretypically achieved through use of an aqueous buffer, such as sodiumbicarbonate, as the solvent.

Suitable solvents are those in which the hGH and the metal cationcomponent are each at least slightly soluble, such as in an aqueoussodium bicarbonate buffer. For aqueous solvents, it is preferred thatwater used be either deionized water or water-for-injection (WFI).

It is understood that the hGH can be in a solid or a dissolved state,prior to being contacted with the metal cation component. It is alsounderstood that the metal cation component can be in a solid or adissolved state, prior to being contacted with the hGH. In a preferredembodiment, a buffered aqueous solution of hGH is mixed with an aqueoussolution of the metal cation component.

Typically, the complexed hGH will be in the form of a cloudyprecipitate, which is suspended in the solvent. However, the complexedhGH can also be in solution. In an even more preferred embodiment, hGHis complexed with Zn⁺².

The complexed hGH is then dried, such as by lyophilization, to form aparticulate of stabilized hGH. The complexed hGH, which is suspended orin solution, can be bulk lyophilized or can be divided into smallervolumes which are then lyophilized. In a preferred embodiment, thecomplexed hGH suspension is micronized, such as by use of an ultrasonicnozzle, and then lyophilized to form stabilized hGH particles.Acceptable means to lyophilize the complexed hGH mixture include thoseknown in the art.

Preferably, particles of stabilized hGH are between about 1 to about 6micrometers in diameter. The hGH particles can be fragmented separately,as described in co-pending U.S. patent application Ser. No. 08/006,682,filed Jan. 21, 1993, which describes a process for producing smallparticles of biologically active agents, which is incorporated herein inits entirety by reference. Alternately, the hGH particles can befragmented after being added to a polymer solution, such as by means ofan ultrasonic probe or ultrasonic nozzle. In another embodiment, asecond metal cation component, which is not contained in the stabilizedhGH particles, is also dispersed within the polymer solution.

It is understood that a second metal cation component and stabilized hGHcan be dispersed into a polymer solution sequentially, in reverse order,intermittently, separately or through concurrent additions. Alternately,a polymer, a second metal cation component and stabilized hGH and can bemixed into a polymer solvent sequentially, in reverse order,intermittently, separately or through concurrent additions.

The method for forming a composition for modulating the release of abiologically active agent from a biodegradable polymer is furtherdescribed in co-pending U.S. patent application Ser. No. 08/237,057.

In this method, the polymer solvent is then solidified to form apolymeric matrix containing a dispersion of stabilized hGH particles.

One suitable method for forming an hGH sustained release compositionfrom a polymer solution is the solvent evaporation method described inU.S. Pat. No. 3,737,337, issued to Schnoring et al., U.S. Pat. No.3,529,906, issued to Vranchen et al., U.S. Pat. No. 3,691,090, issued toKitajima et al., or U.S. Pat. No. 4,389,330, issued to Tice et al.Solvent evaporation is typically used as a method to form hGH sustainedrelease microparticles.

In the solvent evaporation method, a polymer solution containing astabilized hGH particle dispersion, is mixed in or agitated with acontinuous phase, in which the polymer solvent is partially miscible, toform an emulsion. The continuous phase is usually an aqueous solvent.Emulsifiers are often included in the continuous phase to stabilize theemulsion. The polymer solvent is then evaporated over a period ofseveral hours or more, thereby solidifying the polymer to form apolymeric matrix having a dispersion of stabilized hGH particlescontained therein.

A preferred method for forming hGH sustained release microparticles froma polymer solution is described in U.S. Pat. No. 5,019,400, issued toGombotz et al., and co-pending U.S. patent application Ser. No.08/443,726, filed May 18, 1995, the teachings of which are incorporatedherein by reference in their entirety. This method of microsphereformation, as compared to other methods, such as phase separation,additionally reduces the amount of hGH required to produce a controlledrelease composition with a specific hGH content.

In this method, the polymer solution, containing the stabilized hGHparticle dispersion, is processed to create droplets, wherein at least asignificant portion of the droplets contain polymer solution and thestabilized hGH particles. These droplets are then frozen by meanssuitable to form microparticles. Examples of means for processing thepolymer solution dispersion to form droplets include directing thedispersion through an ultrasonic nozzle, pressure nozzle, Rayleigh jet,or by other known means for creating droplets from a solution.

Means suitable for freezing droplets to form microparticles includedirecting the droplets into or near a liquified gas, such as liquidargon and liquid nitrogen to form frozen microdroplets which are thenseparated from the liquid gas. The frozen microdroplets are then exposedto a liquid non-solvent, such as ethanol, or ethanol mixed with hexaneor pentane.

The solvent in the frozen microdroplets is extracted as a solid and/orliquid into the non-solvent to form stabilized hGH containingmicroparticles. Mixing ethanol with other non-solvents, such as hexaneor pentane, can increase the rate of solvent extraction, above thatachieved by ethanol alone, from certain polymers, such aspoly(lactide-co-glycolide) polymers.

A wide range of sizes of hGH sustained release microparticles can bemade by varying the droplet size, for example, by changing theultrasonic nozzle diameter. If very large microparticles are desired,the microparticles can be extruded through a syringe directly into thecold liquid. Increasing the viscosity of the polymer solution can alsoincrease microparticle size. The size of the microparticles can beproduced by this process, for example microparticles ranging fromgreater than about 1000 to about 1 micrometers in diameter.

Yet another method of forming an hGH sustained release composition, froma polymer solution, includes film casting, such as in a mold, to form afilm or a shape. For instance, after putting the polymer solutioncontaining a dispersion of stabilized hGH particles into a mold, thepolymer solvent is then removed by means known in the art, or thetemperature of the polymer solution is reduced, until a film or shape,with a consistent dry weight, is obtained. Film casting of a polymersolution, containing a biologically active agent, is further describedin co-pending U.S. patent application Ser. No. 08/237,057, the teachingsof which are incorporated herein by reference in their entirety.

It is believed that the release of the hGH can occur by two differentmechanisms. The hGH can be released by diffusion through aqueous filledchannels generated in the polymeric matrix, such as by the dissolutionof the hGH or by voids created by the removal of the polymer's solventduring the synthesis of the sustained release composition. A secondmechanism is the release of hGH due to degradation of the polymer.

The rate of degradation can be controlled by changing polymer propertiesthat influence the rate of hydration of the polymer. These propertiesinclude, for instance, the ratio of different monomers, such as lactideand glycolide, comprising a polymer; the use of the L-isomer of amonomer instead of a racemic mixture; and the molecular weight of thepolymer. These properties can affect hydrophilicity and crystallinity,which control the rate of hydration of the polymer. Hydrophilicexcipients such as salts, carbohydrates and surfactants can also beincorporated to increase hydration and which can alter the rate oferosion of the polymer.

By altering the properties of the polymer, the contributions ofdiffusion and/or polymer degradation to hGH release can be controlled.For example, increasing the glycolide content of apoly(lactide-co-glycolide) polymer and decreasing the molecular weightof the polymer can enhance the hydrolysis of the polymer and thus,provides an increased hGH release from polymer erosion.

In addition, the rate of polymer hydrolysis is increased in non-neutralpH's. Therefore, an acidic or a basic excipient can be added to thepolymer solution, used to form the microsphere, to alter the polymererosion rate.

The composition of this invention can be administered to a human, orother animal, by injection, implantation (e.g, subcutaneously,intramuscularly, intraperitoneally, intracranially, intravaginally andintradermally), administration to mucosal membranes (e.g., intranasallyor by means of a suppository), or in situ delivery (e.g. by enema oraerosol spray) to provide the desired dosage of hGH based on the knownparameters for treatment with hGH of the various medical conditions.

The invention will now be further and specifically described by thefollowing examples.

EXAMPLE 1 Formation of Zn⁺² -Stabilized hGH

Human growth hormone (hGH), whose DNA sequence is described in U.S. Pat.No. 4,898,830, issued to Goeddel et al. was used in this Example. Humangrowth hormone was stabilized by forming an insoluble complexes withzinc.

The hGH was dissolved in Samples of a 4 mM sodium bicarbonate buffer (pH7.2) to form hGH solutions with concentrations between 0.1 and 0.5 mMhGH. A 0.9 mM Zn⁺² solution was prepared from deionized water and zincacetate dihydrate and then was added to the hGH solutions to form a Zn⁺²-hGH complex. The pH of the Zn⁺² -hGH solution was then adjusted tobetween 7.0 and 7.4 by adding 1% acetic acid. A cloudy suspendedprecipitate, comprising Zn⁺² -stabilized hGH formed.

The suspension of Zn⁺² -stabilized hGH was then micronized using anultrasonic nozzle (Type V1A; Sonics and Materials, Danbury, Conn.) andsprayed into a polypropylene tub (17 cm diameter and 8 cm deep)containing liquid nitrogen to form frozen particles. The polypropylenetub was then placed into a -80° C. freezer until the liquid nitrogenevaporated. The frozen particles, which contained Zn⁺² -stabilized hGH,were then lyophilized to form Zn⁺² -stabilized hGH particles.

EXAMPLE 2 Preparation of PLGA Microspheres Containing BiologicallyActive, Zn⁺² -Stabilized hGH

Microspheres containing Zn⁺² -stabilized human growth hormone (hGH),were prepared from hydrophilic poly(lactice-co-glycolide) polymer RG502Hhaving free carboxyl end groups (hereinafter "unblocked-PLGA") (50:50PLGA, 9,300 Daltons; Boehringer Ingelheim Chemicals, Inc.) or a morehydrophobic PLGA polymer having blocked carboxyl end groups (hereinafter"blocked-PLGA") (50:50 PLGA, 10,000 Daltons; Lot #115-56-1, BirminghamPolymers, Inc., Birmingham, Ala.).

The polymer was dissolved in methylene chloride at room temperature. Thelyophilized hGH particles were added to the polymer solution and zinccarbonate was also added. The mixture was then sonicated to give ahomogeneous suspension. The suspension was atomized through a sonicatingnozzle on to a bed of frozen ethanol, overlaid with liquid nitrogen. Thevessel containing the microspheres was stored at -80° C. to extract themethylene chloride and then freeze-dried to give a free-flowing powder.

EXAMPLE 3 Analysis of Encapsulated hGH Protein

The integrity of encapsulated hGH was determined by dissolvingunhydrated microspheres into methylene chloride and acetone, collectingthe protein, freeze-drying and re-constituting in HEPES buffercontaining 10 mM EDTA. Appropriate controls were run to ensure that theextraction process did not affect the integrity of the protein.

The integrity of the encapsulated hGH was analyzed by measuring thepercent of hGH monomer contained in the hGH sample after encapsulationby size exclusion chromatography (SEC).

The results of SEC analyses of the hGH integrity of hGH sustainedrelease microspheres are provided below.

    ______________________________________                                        Formulation (polymer; % Zinc Carbonate)                                                             % Monomer (SEC)                                         ______________________________________                                        31K unblocked; 6% ZnCO3                                                                             98.6                                                    31K unblocked; 6% ZnCO3                                                                             99.2                                                    31K unblocked; 3% ZnCO3                                                                             97.7                                                    31K unblocked; 3% ZnCO3                                                                             97.8                                                    31K unblocked; 1% ZnCO3                                                                             97.6                                                    31K unblocked; 0% ZnCO3                                                                             97.8                                                    31K unblocked; 0% ZnCO3                                                                             97.1                                                    10K blocked; 1% ZnCO3 98.2                                                    10K blocked; 1% ZnCO3 98.4                                                    8K unblocked; 0% ZnCO3                                                                              98.5                                                    10K blocked; 1% ZnCO3 98.4                                                    ______________________________________                                    

The results showed that the encapsulation process did not causeaggregation of the protein. The yield percent protein recovered by theextraction procedure (relative to the amount measured by nitrogencontent of the microspheres) ranged from about 40 to 98%.

EXAMPLE 4 Determination of the Effect of Zinc Carbonate on In vitroRelease Kinetics

The microspheres were formed as described in Example 2 and contained 15%w/w hGH (6:1 Zn:hGH protein complex); 0%, 1%, 6%, 10% or 20% w/w zinccarbonate; and poly(lactide-co-glycolide) polymer.

In vitro release kinetics of the hGH sustained release microsphereformulations containing various concentrations of zinc carbonate weredetermined by suspending an aliquot (10 mg) of each type of microspherein different 1.5 ml samples of HEPES buffer (50 mM Hepes, 10 mM KCl 0.1%NaN3) pH 7.2 and then incubating at 37° C. The amount of proteinreleased was quantitated by sampling the buffer at 1, 3, 7, 10, 14, 21,28 days after incubation and replenishing with fresh buffer after eachsampling.

A curve of cumulative percent released (relative to initial hGH contentin the starting mass of microspheres) versus time was plotted. Releasedprotein samples from each time point were assayed for hGH monomercontent by size exclusion chromatography.

Zinc carbonate is thought to act as a reservoir of zinc ions so that theformation of the Zn-hGH complex is favored and dissociation into solublehGH disfavored. Because the aqueous solubility of zinc carbonate is low,the release of zinc ions from the reservoir is slow thus modulating thesolubility of the protein.

In the absence of zinc carbonate, the rate of release of theencapsulated hGH was very rapid and all the protein was released in avery short period.

EXAMPLE 5 Assay for hGH After in Vivo Degradation of Blocked-PLGA Zn⁺²-Stabilized hGh Microspheres

Microspheres of blocked-PLGA, containing 16% w/v Zn⁺² -stabilized hGHand 0%, 6%, 10% or 20% ZnCO₃ were formed by the method of Example 2.Groups of test rats were injected subcutaneously with 50 mg samples ofthe different hGH microspheres. The rats were sacrificed after 60 daysand the skin samples were excised from the injection sites. The excisedskin samples were placed in 10% Neutral Buffered Formalin for at least24 hours. They were then trimmed with a razor blade to remove excessskin and placed in PBS. Tissue samples were processed by PathologyAssociates, Inc. (Frederick, Md.). The skin samples were embedded inglycomethacrylate, sectioned and assayed for the presence of hGH using aHistoScan/LymphoScan Staining Kit (Product #24-408M; Accurate Chemical &Scientific Corp., Westbury, N.Y.) according to the manufacturer'sinstructions. Tissue samples were scored for the presence or absence ofstaining which was indicative of the presence or absence of hGH in thesample. All skin samples, associated with hGH microsphere injections,tested positive for the presence of hGH thus indicating that theblocked-PLGA microspheres still contained hGH after 60 days in vivo.

The method described in Example 2 was used to form microspheres byencapsulating 0% or 15% w/w hGH, in the form of Zn:hGH complex, and also0%, 1% or 6% w/w ZnCO₃ salt, within blocked-PLGA and withinunblocked-PLGA.

In vivo degradation of unblocked-PLGA microspheres versus blocked-PLGAmicrospheres were compared by injecting samples of microspheres intorats and then analyzing the microspheres remaining at the injection siteat various times post-injection. Three rats were assayed at each timepoint for each microsphere sample. On the day of administration of themicrospheres, 750 μl of vehicle (3% carboxymethyl cellulose (lowviscosity) and 1% Tween-20 in saline) was added to vials containing 50±1mg of microspheres. Immediately, the vials were shaken vigorously toform a suspension which was then aspirated into a 1.0 cc syringe withouta needle.

Rats (Sprague-Dawley males) were anesthetized with a halothane andoxygen mixture. The injection sites (intrascapular region) were shavenand marked with a permanent tatoo to provide for the precise excision ofskin at the sampling time points. Each rat was injected with an entirevial of microspheres using 18 to 21 gauge needles.

On designated days (days 15, 30, 59 and 90 post-injection for animalreceiving blocked-PLGA microspheres, or days 7, 14, 21, 28 and 45post-injection for animals receiving unblocked-PLGA microspheres therats were sacrificed by asphyxiation with CO₂ gas and the skin at theinjection sites (including microspheres) was excised. Since themicrospheres tended to clump at the injection sites, the presence orabsence of microspheres was determined visually.

The visual inspections found that the unblocked-PLGA microspheresdegraded substantially faster than the blocked-PLGA microspheres, andthat the addition of ZnCO₃ to the blocked-PLGA substantially slowedpolymeric degradation. For example, in the rats injected withunblocked-PLGA microspheres containing 0% hGH and 0% or 1% ZnCO₃, nomicrospheres were visible on day 21. In addition, for rats injected withblocked-PLGA microspheres containing 0% hGH and 0% ZnCO₃, a fewmicrospheres were visible on day 60 and none were visible on day 90.Furthermore, for rats injected with blocked-PLGA microspheres containing0% or 15% hGH and 6% ZnCO₃, microspheres were visible on day 90.

EXAMPLE 6 In Vivo Pharmacokinetic Studies of hGH Sustained ReleaseMicrospheres in Rats

Studies were conducted in rats to screen various hGH microsphereformulations, determine pharmacokinetic parameters following intravenous(IV), subcutaneous (SC) and SC osmotic pump (Alzet) administration ofhGH, and to evaluate serum profiles and in vivo release rate various hGHmicrosphere formulations.

Sprague-Dawley rats were divided into groups of three each, randomizedby body weight, and one hGH microsphere formulation was administered toeach group. Rats were injected subcutaneously with approximately 7.5 mghGH in 50 mg of microspheres, suspended in 0.75 ml of an aqueousinjection vehicle. The vehicle composition was 3% CMC (low viscosity), 1Polysorbate 20, in 0.9% NaCl. The microsphere dose delivered wasdetermined indirectly by weighing the residual dose in the injectionvial and correcting for residual injection vehicle. The hGH dose wasthen computed from the protein loading of the microspheres determined bynitrogen analysis.

Blood samples were collected at pre-determined intervals for up to 10days after injection. Blood samples of 250 μl were collected during thefirst 24 hours and at least 400 μl at time points after 24 hours. Bloodsamples were clotted and hGH concentrations in serum were determinedusing a radio-immuno assay (RIA) kit from ICN.

For the determination of pharmacokinetic parameters, hGH in saline wasadministered to rats by subcutaneous bolus injection, intravenously ordelivered via an osmotic pump (Alzet Model 2ML4) which was implantedsubcutaneously.

Three groups of rats received single subcutaneous injections of hGH in0.9% NaCl at 0.5 or 7.5 mg/kg at a dose volume of 1.0 ml/kg and twogroups received single intravenous bolus injections of hGH in 0.9% NaClsolution at about 1.0 mg and 5.0 mg of hGH per kg rat with a dose volumeof 1.0 ml/kg. For the Alzet pump study, rats were divided into fourgroups of three rats each, randomized by body weight and dosed withabout 20 mg/ml and 40 mg/ml hGH in 0.9% saline solution loaded intopumps (Alzet Model 2002, 200 μl, 14 days release), and with about 4mg/ml and 12 mg/ml hGH in 0.9% saline solution loaded into pumps (AlzetModel 2ML4, 2 ml, 28 days release). Expected release rates from thepumps correspond to about 2% and 4 to 6% of the dose (about 15 mg/kg)per day, respectively. The Alzet pumps were implanted subcutaneously inthe inter-scapular region after soaking for 1-2 minutes in sterilesaline.

The formulations of hGH sustained release microspheres, synthesized asdescribed in Example 2 contained 15% w/w hGH complexed with Zn in aratio of 6:1 Zn:hGH; 0%, 1%, 3% or 6% w/w zinc carbonate; and 8Kunblocked PLGA, 10K blocked PLGA or 31K unblocked PLGA. To evaluate thevarious hGH sustained release formulations, Cmax, Cd5 and Cmax/Cd5 werethe in vivo indices used, where Cmax is the maximum serum concentrationobserved, and Cd5 is the serum concentration at day 5 which shouldapproximate the steady state concentration. The results were as follows:

    __________________________________________________________________________                        %                                                                        `Burat` in                                                                         Monomer      C day 5                                      Formulation    vitro (%)                                                                          Day 7                                                                              Cmax (ng/ml)                                                                          (ng/ml)                                                                             Cmas/Css                               __________________________________________________________________________    8K unblocked PLGA 0% ZnCO3                                                                   22.0 ± 0.9                                                                       99.3*                                                                             323.3 ± 98.6                                                                       20.4 ± 14.2                                                                      19.5 ± 10.6                         8K unblocked PLGA 1% ZnCO3                                                                   16.4 ± 1.6                                                                       97.3*                                                                             309.0 ± 67.1                                                                       20.4 ± 14.2                                                                      39.5 ± 17.7                         8K unblocked PLGA 3% ZnCO3                                                                   15.9 ± 6.9                                                                      98.7  670.5 ± 244.4                                                                     9.0 ± 4.2                                                                        44.8 ± 22.6                         8K unblocked PLGA 6% ZnCO3                                                                   17.6 ± 2.7                                                                      99.3 358.0 ± 58.9                                                                       18.8 ± 14.7                                                                      42.4 ± 6.8                          31K unblocked PLGA 0% ZnCO3                                                                  12.3 ± 1.1                                                                      98.2   592 ± 318.2                                                                      4.5 ± 1.5                                                                        132.5 ± 47.9                        31K unblocked PLGA 1% ZnCO3                                                                  11.4 ± 1.3                                                                      98.8 432.7 ± 91.6                                                                       5.1 ± 0.3                                                                        84.1 ± 14.9                         31K unblocked PLGA 3% ZnCO3                                                                   7.9 ± 1.9                                                                      99.4  643.6 ± 203.9                                                                     8.0 ± 2.6                                                                        93.3 ± 62.0                         31K unblocked PLGA 6% ZnCO3                                                                  15.8 ± 0.5                                                                      99.8  1691.8 6.6 ± 0.8                                                                        262.2 ± 83.5                        10K unblocked PLGA 1% ZnCO3                                                                  12.7 ± 0.1                                                                      99.3  615.9 ± 384.3                                                                     4.5 ± 1.0                                                                        155.0 ± 126.8                       10K blocked PLGA 3% ZnCO3                                                                    18.1 ± 3.2                                                                      99.6 1053.2 ± 293.3                                                                     3.6 ± 0.8                                                                        291.7 ± 71.1                        10K blocked PLGA 6% ZnCO3                                                                     9.9 ± 1.4                                                                      99.0 1743.5 ± 428.4                                                                     4.9 ± 2.7                                                                        516.1 ± 361.6                       __________________________________________________________________________     *Value obtained from duplicate batch of the same formulation.            

The results of the screening showed that the two unblocked polymers 8Kand 31K had different in vivo release kinetics compared to the originalformulation. Which used blocked 10K PLGA and 6% zinc carbonate. Cmaxvalues were generally lower with the unblocked polymer formulations thanwith the original formulation which suggested that the in vivo `burst`may be lower with the unblocked polymer formulations. The `burst` wasdefined as the percent of hGH released in the first 24 hours afterinjection. The in vitro `burst` values were between 8-22%. The zinccorbonate content of the formulations did not appear to have an effecton the `burst` or the in vitro release profile. The serum concentrationsbetween days 4 and 6 were maintained at a fairly constant level abovebaseline (or the pre-bleed levels) with the unblocked polymerformulations, while serum concentrations with the blocked formulations,at the same time points were close to the baseline levels. The in vitrorelease data for up to 7 days showed that the released hGH protein wasmonomeric. Useful data could not be obtained beyond day 6 because ofanti-hGH antibody formulation in the rats.

EXAMPLE 7 Rhesus Monkey Pharmacokinetics Study

The objective of this primate study was to evaluate the pharmacokineticprofiles of different hGH sustained release formulations as compared tomore traditional methods of administering hGH (e.g., bolus scinjections, daily sc injections and sc injection combined with the useof an osmotic pump) and to determine which hGH sustained releaseformulatin gave the optimal hGH blood concentration profile.

The formulations for the hGH sustained release microspheres testedwere 1) 15% hGH (complexed with Zn at a 6:1 Zn:hGH ratio), 6% w/w zinccarbonate and 10K blocked PLGA; 2) 15% hGH (complexed with Zn at a 6:1Zn:hGH ratio), 1% w/w zinc carbonate and 8K unblocked PLGA ("RG502H"PLGA polymer); and 3) 15% hGH (complexed with Zn at a 6:1 Zn:hGH ratio),1% w/w zinc carbonate and 31K unblocked PLGA ("RG503H" PLGA polymer).

There were four monkeys per group and each animal received a singlesubcutaneous injection into the dorsal cervical region on Day 1. A doseof 160 mg of hGH sustained release microspheres (24 mg of hGH) wasadministered to each monkey in 1.2 ml of injection vehicle through a 20gauge needle. The injection vehicle was an aqueous vehicle containing 3%w/v Carboxymethyl Cellulose (sodium salt), 1% v/v Tween 20 (Polysorbate20) and 0.9% sodium chloride.

The hGH dose was intended to provide measurable hGH serum concentrationsfor pharmacokinetic analysis. To obtain pharmacokinetic parametersadditional study groups of four monkeys each were included,specifically 1) a single subcutaneous injection (24 mg hGH), 2) dailysubcutaneous injections (24 mg/28 days=0.86 mg hGH/day), 3) asubcutaneous injection (3.6 mg hGH) combined with an Alzet osmotic pump(20.4 mg hGH)(total dose of 24 mg hGH), and 4) a subcutaneous injectionof the injection vehicle as a control (only used 3 monkeys for thevehicle control group).

Blood samples were collected at the following times for hGH, IGF1 andanti-hGH antibody analyses: -7, -5, -3 days pre-dose and, 0.5, 1, 2, 3,5, 8, 10, 12, 24, 28, 32 and 48 hours, 5, 4, 6, 8, 11, 14, 17, 20, 23,26, 29, 32, 25, 28, 41, 44, 47, 50, 53, 56 days post-dose.

The concentrations of IGF-1 and hGH in the serum were then measured. AnIRMA kit from RADIM (distributed by: Wein Laboratories, P.O. Box 227,Succasunna, N.J.) was used to quantify hGH in monkey serum. The IRMAassay had a limit of quantification in PBS buffer of 0.1 ng/mL and inpooled juvenile rhesus monkey serum of 1.5 ng/mL with a basal GH levelof about 4 ng/mL.

The results showed that the hGH sustained release microspheres werereleasing significant, sustained levels of hGH over a one month periodwhile the subcutaneous injections were not able to maintain the sameserum levels.

The IGF-1 serum profile showed that serum IGF-1 concentrations wereelevated above the baseline values between days 2 and 29 afteradministering the microparticles. This shows that enough hGH was beingreleased from the hGH sustained release microspheres to cause apharmacodynamic effect. This also indicates that the hGH released wasbiologically active which suggest that the encapsulation process had notadversely affected the biopotency of hGH.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

The invention claimed is:
 1. A composition for the sustained release ofhuman growth hormone from a polymeric matrix, comprising:a) abiocompatible polymer; and b) particles of metal cation-complexed humangrowth hormone, wherein said particles are dispersed within thebiocompatible polymer.
 2. A sustained release composition of claim 1wherein the biocompatible polymer is selected from the group consistingof poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polycyanoacrylates,poly(p-dioxanone), poly(alkylene oxalate)s, biodegradable polyurethanes,blends and copolymers thereof.
 3. A sustained release composition ofclaim 2 wherein said polymer comprises poly (lactide-co-glycolide).
 4. Asustained release composition of claim 1 wherein the metalcation-complexed human growth hormone contains a multivalent metalcation component.
 5. A sustained release composition of claim 4 whereinthe metal cation component contains Zn⁺².
 6. A sustained releasecomposition of claim 1 further comprising a second metal cationcomponent, wherein the second metal cation component is not complexed tosaid human growth hormone, and wherein the second metal cation componentis dispersed within the biocompatible polymer to modulate the release ofhuman growth hormone from the polymeric matrix.
 7. A composition for thesustained release of human growth hormone from a polymeric matrix,comprising:a) poly(lactide-co-glycolide); b) particles of zinc-complexedhuman growth hormone, with a zinc-to-human growth hormone molar ratiobetween 4:1 and 100:1, wherein said particles are dispersed within thepoly(lactide-co-glycolide, and wherein the proportion of human growthhormone in the sustained release composition is between 10 and 30 weightpercent; and c) a second metal cation component, wherein the secondmetal cation component is not complexed to said human growth hormone,and wherein the second metal cation component is dispersed within thepolymeric matrix at a concentration of at least about 1% by weight ofpolymer.
 8. A sustained release composition of claim 7 wherein theparticles of zinc-complexed human growth hormone also contain sodiumbicarbonate.
 9. A method for providing a therapeutically effective bloodlevel of biologically active, non-aggregated human growth hormone in asubject for a sustained period, comprising administering to the subjecta dose of the sustained release composition of claim
 1. 10. A sustainedrelease composition of claim 7 wherein said second metal cationcomponent is zinc carbonate.
 11. A sustained release composition ofclaim 10 wherein said zinc carbonate is dispersed within the polymericmatrix at a concentration of at least about 3% by weight of polymer.